Recombinant human osteogenic protein-1 induces bone formation in a chronically infected, internally stabilized segmental defect in the rat femur

BACKGROUND: Recombinant human osteogenic protein-1 (rhOP-1), combined with a collagen carrier, has been shown to induce new-bone formation in a variety of animal models. The purpose of the present investigation was to test the hypotheses that rhOP-1 would accelerate bone formation in an internally stabilized, chronically infected, critical-size defect in the rat femur and that this effect would be enhanced by the administration of systemic antibiotic. METHODS: A 6-mm segmental defect was created surgically, stabilized with a polyacetyl plate and six Kirschner wires, and contaminated with 10(4) colony-forming units of Staphylococcus aureus in one femur in each of 168 Sprague-Dawley rats. After two weeks, these infected defects were débrided surgically and were assigned to one of six treatment groups. The defects in the thirty animals in the first group received lyophilized collagen carrier mixed with 200 microg of rhOP-1 dissolved in buffer, the defects in the thirty animals in the second group received carrier with 20 microg of rhOP-1 in buffer, and the defects in the twenty-four control animals in the third group received carrier mixed with buffer without rhOP-1. The last three groups were treated identically to the first three groups, except that the animals also received the antibiotic ceftriaxone for twenty-eight days after débridement. The animals were killed at two, four, eight, or twelve weeks after débridement. Newly mineralized callus within the defect, and adjacent to and bridging the outside of the defect, was assessed with use of quantitative high-resolution radiography, microcomputed tomography, torsional failure testing, and histological analysis of undecalcified sections. RESULTS: Bacterial cultures confirmed the presence of a chronic infection during the study period in all animals. At the later time-points, significantly more newly mineralized callus was present within and adjacent to the débrided defects that had been treated with 200 microg of rhOP-1, whereas minimal amounts of callus were present within and adjacent to the defects that had been treated without rhOP-1 and with 20 microg of rhOP-1. At eight and twelve weeks after débridement, there was significantly more newly mineralized callus in the group that had been treated with 200 microg of rhOP-1 with antibiotic than in the group that had been treated with 200 microg of rhOP-1 without antibiotic (p < 0.05). At twelve weeks, the values for torque, energy to failure, and linear stiffness for femora that had been treated with 200 microg of rhOP-1 with antibiotic were not significantly different from the values for intact, contralateral control femora, whereas the values for femora that had been treated with 200 microg of rhOP-1 without antibiotic remained significantly lower than those for the intact, contralateral controls (p < 0.05). CONCLUSIONS: Recombinant human osteogenic protein-1 maintained its osteoinductive capability in the presence of chronic infection, and this property was enhanced by antibiotic therapy. No substantial callus formed in the infected defects without a sufficiently high dose of rhOP-1. CLINICAL RELEVANCE: The treatment of an infection at the site of a fracture often necessitates removal of internal fixation. However, internal fixation is needed for fracture stability. This study presents an intervention that may accelerate fracture-healing in the presence of infection and colonized hardware, thereby permitting earlier removal of the hardware and more timely and effective treatment of the infection.     

Osteogenic protein-1 induced bone formation in an infected segmental defect in the rat femur.

The goal of this study was to use a segmental defect model in the rat femur to determine if osteogenic protein-1 (OP-1) is capable of inducing bone formation in the presence of bacterial contamination. A 6 mm segmental defect was surgically created and stabilized with a polyacetyl plate and Kirschner wires in one femur in each of 126 Sprague-Dawley rats. The animals were divided into eight groups in which the defect was either left untreated, or subjected to various combinations of OP-1 (11 or 50 microg), lyophilized bovine type I collagen (carrier for the OP-1), and 10(5) colony-forming units of Staphylococcus aureus. The animals were euthanized at either 2, 4, or 9 weeks. Quantitative radiographic and histologic analyses were performed on the harvested tissue. The initial contamination progressed to infection in all animals receiving bacteria, as determined by qualitative bacteriology. There was very little, if any, bone formation in the untreated defects, and in the contaminated defects with or without collagen carrier. Bone formation was significantly greater in contaminated defects with either dose of OP-1, compared with contaminated defects without OP-1. The 50 microg dose of OP-1 induced significantly more bone formation than the 11 microg dose, both with and without bacteria. This investigation has demonstrated that OP-1 maintains its osteoinductive capability in a contaminated segmental defect. OP-1 may potentially be used in the clinical management of contaminated fractures.     

Characterization of a chronic infection in an internally-stabilized segmental defect in the rat femur.

The aim of this study was to characterize a new model of chronic osteomyelitis with clinically relevant features. A segmental defect of critical size was surgically created in the rat femur, stabilized with a polyacetyl plate and Kirschner wires, and contaminated with bacteria. The animals were allowed to recover while the contamination progressed to a chronic infection. At a later point in time, the defect was surgically débrided without removing the implant. Further treatments of interest, such as antibiotic therapy or application of an osteogenic agent, could be introduced at this time. To implement this model, an initial experiment was performed to determine the bacterial inoculum and time from contamination that would reliably result in an infected defect without causing excessive bone damage by the time débridement surgery was performed. The number of recovered bacteria, degree of radiographic bony lysis, and torsional stiffness of the defect fixation were measured in 192 rats as a function of 4 inocula of Staphylococcus aureus (10(3), 10(4), 10(5) or 10(6) CFUs) and 4 times from contamination (1, 2, 3 or 4 weeks). A 10(4) CFU inoculum over 2 weeks was found to consistently create an infection without severe lysis and loss of fixation stability. Based on these values, a second experiment was performed in 96 rats to characterize the débrided defect over time (2, 4, 8 and 12 weeks after débridement), with and without 4 weeks of the antibiotic ceftriaxone, in terms of the same outcome variables. Infection was persistent in all animals in spite of débridement and antibiotic therapy. Antibiotic therapy did not reduce the degree of bony lysis. Compared with animals not given antibiotic, bacterial counts significantly decreased during the period of antibiotic therapy, but then rebounded to significantly higher levels at 12 weeks. This model allows us to perform further studies on differing regimens of antibiotic therapy and their relationship to surgical débridement, and on the efficacy of osteogenic agents in the presence of infection.     

Comparison of two different fixation techniques for a segmental defect in a rat femur model.

Studies have attempted to identify the osteogenic effects of bone morphogenetic proteins using a rat femur model, which commonly involves the creation of a critical size defect followed by internal fixation of the femur. Among the most familiar fixation methods are either plating or intramedullary placement of a Kirschner wire (K-wire). There are advantages and disadvantages to each method; however, this study attempts to identify the best method by exploring the histological effects of each technique. The experiment involved two groups with no added treatment: Group P (plate fixation method) and Group K (K-wire fixation method). The animals were allowed a 4-week interval for the femurs to heal, and proximal, distal, and two midshaft cuts were examined under high-power microscopy after the fixation apparatus was removed. Group K exhibited a peculiar fibrotic healing pattern that followed the shaft of the then vacated K-wire and there was minimal new viable bone formation. Group P, however, exhibited a more natural ingrowth of newly formed bone that began at the proximal and distal cuts and proceeded centrally into the core of the defect. Due to the fibrotic tissue in Group K, this study shows that the model is insufficient due to the micromotion created and thus supports plating of critical defects as the fixation method of choice due to the creation of a stable healing environment.     

Interaction between living bone particles and rhBMP‐2 in large segmental defect healing in the rat femur

Orthopedic surgeons sometimes combine recombinant, human BMP‐2 with autograft bone when dealing with problematic osseous fractures. Although some case reports indicate success with this off‐label strategy, there have been no randomized controlled trials. Moreover, a literature search revealed only one pre‐clinical study and this was in a cranial defect model. The present project examined the consequences of combining BMP‐2 with particles of living bone in a rat femoral defect model. Human bone particles were recovered with a reamer‐irrigator‐aspirator (RIA). To allow acceptance of the xenograft as surrogate autograft, rats were administered an immunosuppressive cocktail that does not interfere with bone healing. Implantation of 200 µg living bone particles generated a small amount of new bone and defects did not heal. Graded amounts of BMP‐2 that alone provoked no healing (1.1 µg), borderline healing (5.5 µg), or full healing (11 µg) were added to this amount of bone particles. Addition of BMP‐2 (1.1 µg) increased osteogenesis, and produced bridging in 2 of 7 defects. The combination of BMP‐2 (5.5 µg) and bone particles made healing more reliable and advanced the maturation of the regenerate. Bone formation with BMP‐2 (11 µg) and bone particles showed improved maturation. Thus, the combination of autograft and BMP‐2 may be helpful clinically under conditions where the healing response is suboptimal. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:2137–2145, 2016. Clinical significance These data support the clinical use of recombinant, human BMP‐2 with autograft bone when treating large segmental osseous defects. The combination leads to greater bone formation and accelerates the maturation of the regenerate.     

Estimation of defect volume in segmental defects of the tibia and femur

BACKGROUND: With the advent of modern limb salvage techniques, segmental bone loss in the lower extremity has become more common. METHODS: To aid preoperative planning when dealing with segmental bone loss in the femur and tibia, we performed a cadaveric study to estimate the volume of autogenous or allograft material required to fill defects located in various areas of the bones. RESULTS: The greatest volume was generally required in metaphyseal defects, with an average of 12 cc/cm in the distal femur and proximal tibia, 11 cc/cm in the proximal femur, and 6 cc/cm in the distal tibia. Diaphyseal defects were found to have the least variability with regard to the volume of graft material required for different specimens. Femoral diaphyseal defects required 7 cc/cm and tibial diaphyseal defects required 5 cc/cm. A slightly larger volume of allograft material was needed to fill all defects compared with autograft. CONCLUSION: This method allows one to estimate the amount of graft required for a defect of the femur and the tibia.     

Non-union site debridement increased the efficacy of rhBMP-2 in a rodent model

In our study we investigated the influence of debridement on bone healing in a rodent critical size defect model with and without rhBMP-2 in fibrin matrix.     

Treatment of a double nonunion of the femur by rhBMP-2

A 49-year-old patient sustained an ipsilateral fracture of the lateral femoral neck and distal femoral shaft, which were treated with a long intramedullary nail with a hip screw component. Both fractures did not heal, and both nonunion sites were revised by reosteosynthesis with a dynamic hip screw for the femoral neck and a locked intramedullary nail for the shaft region combined with autogenous bone grafting at both sites. At 14 months from injury and after 2 operations, both nonunions persisted. At a third surgery, 1 kit of recombinant human bone morphogenetic protein-2 (rhBMP-2) on an absorbable collagen sponge was applied to each site, without any modification of the osteosynthesis or additional bone grafting. The lateral femoral neck and the femoral shaft consolidated 24 and 30 weeks, respectively, after the rhBMP-2 application, and the patient resumed his work as industrial worker after 7 months after his last surgery. We believe this is the first study to report the successful use of 2 kits of rhBMP-2 in a double nonunion of the femur.     

Long-term follow-up study of bioactive bone cement for repairing a segmental defect in a canine femur

We report on a 7-year long-term follow-up study of a bioactive bone cement (BA cement) that was used to repair a segmental defect in a canine femur. Bilateral femoral segmental defects were repaired with metallic implants that were fixed to the femur using two kinds of bone cement. The BA cement used in this study consists of an apatite- and wollastonite-containing glass ceramic (AW-GC) with a bis-phenol-alpha-glycidyl methacrylate (bis-GMA)-based resin. The bone-cement interface was examined histologically. Previous short-term studies have shown that using BA cement for segmental replacement of the canine femur produced excellent biomechanical and histological results. The BA cement maintained the fixation of a metallic implant to the femur very well. In contrast, the PMMA cement did not maintain alignment under long-term weight-bearing conditions. The results of histological examinations showed direct bonding between the BA cement and bone, while an intervening soft tissue layer was observed at the bone-cement interface with the PMMA cement. The BA cement bonded to the bone through a Ca-P-rich reactive layer, which was twice as thick after 7 years than it was at 26 weeks. No adverse effects of BA cement were observed during the 7-year observation period.     

Increased bone formation in a rabbit long-bone defect model after single local and single systemic application of erythropoietin

Background and purpose — Delayed bone healing with non-union is a common problem. Further options to increase bone healing together with surgery are needed. We therefore evaluated a 1-dose single application of erythropoietin (EPO), applied either locally to the defect or systemically during surgery, in a critical-size rabbit long-bone defect.

Material and methods — 19 New Zealand White rabbits received a 15-mm defect in the radius diaphysis. An absorbable gelatin sponge was soaked with saline (control group and systemic treatment group) or EPO (local treatment group) and implanted into the gap. The systemic treatment group received EPO subcutaneously. In vivo micro-CT analysis was performed 4, 8, and 12 weeks postoperatively. Vascularization was evaluated histologically.

Results — Semiquantitative histomorphometric and radiological evaluation showed increased bone formation (2.3- to 2.5-fold) in both treatment groups after 12 weeks compared to the controls. Quantitative determination of bone volume and tissue volume showed superior bone healing after EPO treatment at all follow-up time points, with the highest values after 12 weeks in locally treated animals (3.0- to 3.4-fold). More vascularization was found in both EPO treatment groups.

Interpretation — Initial single dosing with EPO was sufficient to increase bone healing substantially after 12 weeks of follow-up. Local application inside the defect was most effective, and it can be administered directly during surgery. Apart from effects on ossification, systemic and local EPO treatment leads to increased callus vascularization.     

Histomorphometric description of allograft bone remodeling and union in a canine segmental femoral defect model: a comparison of rhBMP-2, cancellous bone graft, and absorbable collagen sponge.

The purpose of this study was to determine the effect of recombinant human bone morphogenetic protein type 2 (rhBMP-2) on the histomorphometry of femoral allograft-host bone union and allograft remodeling. A 6 cm mid-diaphyseal femoral defect was created and filled with an allograft stabilized with an interlocking nail in 21 dogs. Dogs were randomly divided into three equal groups and the allograft-host bone junctions and the mid-diaphyses of the allografts were treated with either an absorbable collagen sponge (ACS) loaded with rhBMP-2 (BMP group), an autogenous cancellous bone graft (CBG group), or ACS loaded with buffer solution (ACS group). All dogs received daily tetracycline until sacrifice at 24 weeks to label new bone formation. Histomorphometric analyses on sections of proximal and distal allograft-host bone junctions and the mid-diaphyseal portion of allografts were performed using fluorescent and regular light microscopy. Analyses of the host bone and junctions between allograft and host bone revealed significantly greater new bone formation and larger osteon radii in the BMP group compared to CBG and ACS groups and contralateral intact bone. Porosity in CBG and ACS groups was significantly higher than in the BMP group, which had similar values to intact bone. In transverse sections of allografts, the largest pore diameters were present in the CBG group. Based on all parameters measured, significantly higher bone turnover occurred in the outer cortical area of the allograft in all groups as compared to the inner cortical and mid-cortical areas. New bone formation and osteon radius/osteon width in allografts were similar for all three groups. Higher porosity and larger pore diameters in the CBG and ACS groups suggested higher bone resorption versus formation in these groups compared to the BMP group. The results of this study reveal more balanced allograft bone resorption and bone formation in the BMP group, with greater resorptive activity in the CBG and ACS groups. However, neither rhBMP-2 nor autogenous bone graft increased allograft incorporation when compared to the negative control (ACS group).     

Effect of low molecular weight heparin on fracture healing in a stabilized rat femur fracture model.

The purpose of this study was to evaluate the effect of low molecular weight heparin (LMWH) on fracture healing in a standard stabilized rat femur fracture model. A closed, mid-diaphyseal transverse fracture was created in the right femur of Long-Evans rats after insertion of a 0.8-mm K-wire into the medullary canal. Animals were randomized to receive either LMWH (70 units/kg dalteparin) or an injection of normal saline daily for 2 weeks. Animals were sacrificed at 2, 3, and 6 weeks. Fracture healing was assessed by radiographs, histology, and mechanical testing. There were no significant differences between the control and LMWH groups in the percentage of animals with radiographic bridging callus at each time point. Histologic appearance of fracture healing was similar between the control and LMWH groups. There were no significant differences in the normalized mechanical properties of the control and LMWH groups at 2 and 3 weeks. At 6 weeks, the percent torque of the LMWH group was significantly greater than the control group ( p = 0.0072), however, there was no significant difference in the stiffness and energy absorption. Dalteparin, at the dosage used in this study, did not impair fracture healing in this standard stabilized rat femur fracture model.     

仿生活性人工骨修复兔桡骨节段性骨缺损的研究

目的 研制在成分和结构上与天然骨基质高度仿生的新型材料，应用于节段性骨缺损的修复。方法 以Ⅰ型胶原蛋白分子为模板，引入钙磷盐生成体重，合成纳米相羟基磷灰石/胶原（NHAC）并以之为主体，以1：1的质量比加入聚乳酸（PLA）制备多孔框架，与重组人骨形态发生蛋白2（rh-BMP2)复合后植入兔桡骨15mm缺损，观察骨修复情况。结果 所合成的材料具有纳米级的层装排列结构。层间距为11.7nm，实验组中10只兔术后12周骨缺损均完全愈合。结论 NHAC可能成为修复骨缺损的较理想材料，具有较大的临床应用潜力。     

rhBMP2与rhVEGF联合用药对大鼠成骨细胞及跖骨的作用

目的 观察rhBMP2（重组人骨形成蛋白-2）与rhVEGF（重组人表皮生长因子）联合用药对体外培养SD大鼠成骨细胞不同成熟阶段的增殖、分化及胎鼠跖骨生长的影响。方法分离新生大鼠颅盖骨的细胞培养获得前成骨细胞（preosteoblast,pOB）,再诱导分化为成骨细胞（osteoblast,OB）,以rhBMP2和低(2?g/L）、中(4?g/L)及高(8?g/L)剂量rhVEGF干预48h,比较乳酸脱氢酶（LDH）活性、细胞相对增殖率（RGR）和碱性磷酸酶（AKP）活性。另取胎鼠跖骨,以rhBMP2和rhVEGF干预7d和14d,比较其纵向骨生长率（longitudinal bone growth rate,LBDR）。结果 ①LDH组间差异无统计学意义。②RGR：pOB的中、高剂量联用组较同剂量rhBMP2组高。OB的中、高剂量联用组较同剂量rhBMP2及rhVEGF组高。③AKP：pOB的中、高剂量联用组较同剂量rhBMP2及rhVEGF组高。OB的组间差异无统计学意义。④LBDR;7d联用组较rhBMP2及rhVEGF组高,但14d联用组差异无统计学意义。结论 适量联用rhBMP2与rhVEGF对骨再生诱导及骨组织早期生长有协同促进效应。     

Substance P and prostaglandin E2 release after shock wave application to the rabbit femur

The biologic action of extracorporeal shock wave application on the musculoskeletal system is poorly understood. To prove the hypothesis that alterations of tissue concentrations of substance P and prostaglandin E(2) are involved in the biologic action of shock waves, extracorporeal shock waves with energy flux density of 0.9 mJ/mm2 (1500 pulses at 1/second) were applied in vivo to the distal femur of rabbits. The concentrations of substance P and prostaglandin E(2) eluted from the periosteum of the femur were measured. Compared with the untreated contralateral hindlimbs, substance P release from the periosteum from the femur was increased 6 hours and 24 hours after extracorporeal shock wave application, but was decreased 6 weeks after extracorporeal shock wave application. By contrast, extracorporeal shock wave application did not result in altered prostaglandin E(2) release from the periosteum from the femur. Remarkably, there was a close relationship between the time course of substance P release found here, and the well-known clinical time course of initial pain occurrence and subsequent pain relief after extracorporeal shock wave application to tendon diseases. Accordingly, substance P might be involved in the biologic action of extracorporeal shock wave application on tissue of the musculoskeletal system. This is the first study providing insights into the molecular mechanisms of extracorporeal shock wave application to the musculoskeletal system.     

The induced membrane technique: Optimization of bone grafting in a rat model of segmental bone defect

IntroductionThe induced membrane technique (IMT) is a two-stage surgical procedure used to treat fracture nonunion and bone defects. Although there is an increasing number of animal studies investigating the IMT, few have examined the outcomes of bone healing after a second stage grafting procedure. This study aimed at comparing two bone grafting procedures, as part of the IMT, in order to establish a rat model providing consistent healing outcomes.MethodsIn male Fischer 344 rats, we created a 5 mm defect in the right femur, stabilized the bone with a plate and screws, and inserted a polymethylmethacrylate spacer into the defect. Four weeks later, the spacer was removed. Bone graft was harvested from a donor rat and placed into the defect, followed by membrane and wound closure. Experiments were conducted in two groups. In group 1 (n = 11), the bone graft contained a variable amount of cortical and cancellous bone, the time from donor euthanasia to grafting was up to 240 min, and one donor rat provided graft for 5-6 recipients. In group 2 (n = 12), we reduced the contribution of cortical bone to the graft, included bone marrow, and kept donor euthanasia to grafting time under 150 min. One donor was used per 3-4 recipients. The volume of graft per recipient and all other elements of the protocol were the same across groups. Bone healing at 12 weeks post grafting was compared radiographically by two orthopaedic surgeons in a blinded fashion, based on union status and a modified Lane & Sandhu score.ResultsHealing rates improved from 36.4% in Group 1 to 91.6% in Group 2. There was a significant relationship between the methods and resulting union status (p = 0.004). The odds of achieving full union were significantly higher in group 2 compared to group 1 (odds ratio=19.25, 95% confidence interval [1.77-209.55]; p = 0.009). The average radiographic score was also significantly higher in group 2 (p = 0.005).ConclusionThe revised bone grafting method significantly improved the healing outcomes and contributed to establishing a consistent rat model of the IMT. This model can benefit preclinical investigations by allowing for reliable and clinically-relevant comparisons.